Application of ryr2 protein or recombinant ryr2 protein in preparing anti-heart failure medicament

ABSTRACT

An application of a RyR2 protein or a recombinant RyR2 protein in preparing an anti-heart failure medicament is provided. The recombinant RyR2 protein is a naturally occurring RyR2 protein fragment or a mutant, such as a SPRY1 domain protein, a P1 domain protein, a SPRY2 domain protein, a SPRY3 domain protein, a Handle domain protein, an HD1 domain protein, an HD2 domain protein, a central domain protein, an EF-hand domain protein, a U-motif protein, a P2 domain protein, a P2 domain fragment protein-1, a P2 domain fragment protein-2 derived from a natural RyR2 protein or a P2 mutant derived from the natural RyR2 protein. The exogenous recombinant RyR2 protein is highly expressed in both normal small animal and diseased small animal models, so that the left ventricular ejection fraction of experimental animals is improved varying degrees compared with that of a control group.

CROSS REFERENCE TO THE RELATED APPLICATIONS

This application is the national phase entry of International Application No. PCT/CN2019/095015, filed on Jul. 8, 2019, which is based upon and claims priority to Chinese Patent Application No. 201810744453.3, filed on Jul. 9, 2018, the entire contents of which are incorporated herein by reference.

TECHNICAL FIELD

The present invention relates to the technical field of biomedical engineering, and in particular to a recombinant ryanodine receptor type 2 (RyR2) protein used for enhancing contractility of cardiomyocytes, a polynucleotide sequence encoding the recombinant RyR2 protein, a vector and a medicament containing the recombinant RyR2 protein, and an application thereof.

BACKGROUND

Heart failure is the final stage of most cardiac diseases, which is one of the main causes contributing to human morbidity and mortality. The incidence rate of heart failure is increasing worldwide. Although the current medicine treatment has played a certain role in controlling the symptoms and mortality of heart failure, it cannot fundamentally reverse the development of the disease. 50% of heart failure patients will die within 5 years. At the cellular level, heart failure is mainly characterized by abnormal contraction and abnormal rhythm of cardiomyocytes.

Muscle tissue, which can be considered as the largest organ of vertebrates, can be divided into skeletal muscle tissue, myocardial tissue and smooth muscle tissue. Both skeletal muscle tissue and myocardial tissue belong to striated muscle tissue and have similar biological regulation effect. For example, skeletal muscle cells and cardiomyocytes have very similar process of excitation-contraction coupling. Membrane depolarization of myocytes and activated L-type voltage-gated calcium channel cause calcium to flow into the cytoplasm (sarcoplasm) of myocytes. The increased calcium concentration in cytoplasm can further activate ryanodine receptors (RyR) through the mechanism of calcium-induced calcium release (CICR). This leads to the release of calcium from sarcoplasmic reticulum (SR), and thus leads to a further rapid increase in calcium concentration of cytoplasm. Calcium ions diffuse through the cytoplasm and bind to actin (part of contractile proteins), such as troponin C, causing myocytes to contract. After contraction, the sarcoplasmic reticulum retrieves calcium ions and finally removes calcium in the cytoplasm mainly through the action of sarcoplasmic/endoplasmic reticulum calcium ATPase (SERCA). These biological processes are basically the same in skeletal muscle cells and cardiomyocytes, but there also exist some minor differences involved in these biological processes. For example, although RyR1 protein is a major release channel of calcium from the sarcoplasmic reticulum in skeletal muscle cells, RyR2 is dominant in cardiomyocytes. Similarly, SERCA in skeletal muscle cells is SERCA1a, while SERCA2a is specific in cardiomyocytes.

The calcium cycling and calcium homeostasis of cardiomyocytes are maintained by several key proteins. The disorder of calcium cycling leads to a variety of myocyte pathological processes, such as cardiac insufficiency, contractile ventricular dysfunction, arrhythmia, heart failure, cardiogenic shock, myocardial infarction and cardiac valve dysfunction. Ryanodine receptor type 2 (RyR2, UniProtKB No. Q92736) is a major calcium release channel in sarcoplasmic reticulum of cardiomyocytes. The amount of calcium ions released through RyR2 determines the amplitude of calcium transients, and the contractility of cardiomyocytes depends on the amplitude of calcium transients. In the pathological process of heart failure, previous studies showed that abnormal modification of RyR2 specific sites could be observed in heart failure patients and disease animal models, such as phosphorylation at residue serine 2808 and residue serine 2814 of RyR2 (Wehrens X H T, Lehnart S E, Reiken S, et al. Ryanodine receptor/calcium release channel PKA phosphorylation: a critical mediator of heart failure progression[J]. Proceedings of the National Academy of Sciences, 2006, 103(3): 511-518; Respress J L, van Oort R J, Li N, et al. Role of RyR2 phosphorylation at 52814 during heart failure progression[J]. Circulation Research, 2012: CIRCRESAHA. 112.268094.). It is generally believed that the abnormal modification of RyR2 specific sites leads to some changes in protein conformation, resulting in the dissociation of a channel-switch protein, FK506-binding protein 12.6 (FKBP12.6), increasing the probability of calcium channel opening, reducing the number of calcium ions released each time, and enhancing the pathological process of calcium leak.

Therefore, it is an urgent scientific challenge to reduce abnormal modification of RyR2 in myocardial pathological process by biomedical means.

SUMMARY

The objective of the present invention is to provide a novel recombinant ryanodine receptor type 2 (RyR2) protein capable of enhancing the contractility of cardiomyocytes, a polynucleotide sequence encoding the recombinant RyR2 protein, a vector and a medicament containing the recombinant RyR2 protein, and an application thereof.

A first aspect of the present invention provides a recombinant RyR2 protein, which is a naturally occurring RyR2 protein fragment or a mutant, and the length of the fragment or mutant is not less than 15 consecutive amino acid residues.

The recombinant RyR2 protein of the present invention is a positive inotropic peptide, which exhibits the ability to enhance the contractility of cardiomyocytes and/or to rebalance the calcium cycling.

In a preferred embodiment of the present invention, the fragment or the mutant may enhance cardiac function, and the fragment or the mutant has at least one function including, but not limited to, antiarrhythmia, antiapoptosis, reducing a spontaneous calcium spark frequency in cardiomyocytes, preventing and/or reducing calcium leak from sarcoplasmic reticulum, and restoring a hemodynamic function in individuals with heart failure.

Preferably, the recombinant RyR2 protein includes a SPRY1 domain protein, a P1 domain protein, a SPRY2 domain protein, a SPRY3 domain protein, a Handle domain protein, an HD1 domain protein, an HD2 domain protein, a central domain protein, an EF-hand domain protein, a U-motif protein, a P2 domain protein, a P2 domain fragment protein-1 and a P2 domain fragment protein-2. These fragments are derived from a natural RyR2 protein.

A second aspect of the present invention provides a gene encoding a recombinant RyR2 protein, preferably, a gene encoding a protein including a SPRY1 domain protein, a P1 domain protein, a SPRY2 domain protein, a SPRY3 domain protein, a Handle domain protein, an HD1 domain protein, an HD2 domain protein, a central domain protein, an EF-hand domain protein, a U-motif protein, a P2 domain protein, a P2 domain fragment protein-1 and a P2 domain fragment protein-2. These fragments are derived from the natural RyR2 protein.

In a preferred embodiment of the present invention, cardiac biological function can be enhanced by using the recombinant RyR2 protein or a polynucleotide encoding the recombinant RyR2 protein. The cardiac biological function is at least one selected from the group consisting of antiarrhythmia, antiapoptosis, reducing a spontaneous calcium spark frequency in cardiomyocytes, preventing and/or reducing calcium leak from sarcoplasmic reticulum, and restoring a hemodynamic function in individuals with heart failure.

Therefore, the recombinant RyR2 protein or the polynucleotide encoding the recombinant RyR2 protein can be used to achieve anti-arrhythmic function on the cardiomyocytes, and therefore, preferably, to protect cardiomyocytes and cardiac tissues from arrhythmias, especially catecholamine-evoked arrhythmias, such as ventricular arrhythmia associated with sudden cardiac death.

Additionally, the recombinant RyR2 protein or the polynucleotide encoding the recombinant RyR2 protein can also protect subjects from lethal ventricular tachyarrhythmias, such as adrenergic receptor-mediated lethal ventricular tachycardias and catecholamine-mediated lethal ventricular tachyarrhythmias.

In a further preferred embodiment, the recombinant RyR2 protein or the polynucleotide encoding the recombinant RyR2 protein also has an ability to reduce a calcium spark frequency in cardiomyocytes.

In a preferred embodiment of the present invention, when the recombinant RyR2 protein is the SPRY1 domain protein derived from the natural RyR2 protein, an amino acid sequence of the protein has at least 60%, preferably at least 65%, preferably at least 70%, more preferably at least 75%, more preferably at least 80%, more preferably at least 85%, even more preferably at least 90%, even more preferably at least 95% and most preferably at least 99% identity to an amino acid sequence shown in SEQ ID NO.1; a polynucleotide sequence encoding the protein has at least 60%, preferably at least 65%, preferably at least 70%, more preferably at least 75%, more preferably at least 80%, more preferably at least 85%, even more preferably at least 90%, even more preferably at least 95% and most preferably at least 99% identity to a polynucleotide sequence shown in SEQ ID NO.2.

In a preferred embodiment of the present invention, when the recombinant RyR2 protein is the P1 domain protein derived from the natural RyR2 protein, an amino acid sequence of the protein has at least 60%, preferably at least 65%, preferably at least 70%, more preferably at least 75%, more preferably at least 80%, more preferably at least 85%, even more preferably at least 90%, even more preferably at least 95% and most preferably at least 99% identity to an amino acid sequence shown in SEQ ID NO.3; a polynucleotide sequence encoding the protein has at least 60%, preferably at least 65%, preferably at least 70%, more preferably at least 75%, more preferably at least 80%, more preferably at least 85%, even more preferably at least 90%, even more preferably at least 95% and most preferably at least 99% identity to a polynucleotide sequence shown in SEQ ID NO.4.

In a preferred embodiment of the present invention, when the recombinant RyR2 protein is the SPRY2 domain protein derived from the natural RyR2 protein, an amino acid sequence of the protein has at least 60%, preferably at least 65%, preferably at least 70%, more preferably at least 75%, more preferably at least 80%, more preferably at least 85%, even more preferably at least 90%, even more preferably at least 95% and most preferably at least 99% identity to an amino acid sequence shown in SEQ ID NO.5; a polynucleotide sequence encoding the protein has at least 60%, preferably at least 65%, preferably at least 70%, more preferably at least 75%, more preferably at least 80%, more preferably at least 85%, even more preferably at least 90%, even more preferably at least 95% and most preferably at least 99% identity to a polynucleotide sequence shown in SEQ ID NO.6.

In a preferred embodiment of the present invention, when the recombinant RyR2 protein is the SPRY3 domain protein derived from the natural RyR2 protein, an amino acid sequence of the protein has at least 60%, preferably at least 65%, preferably at least 70%, more preferably at least 75%, more preferably at least 80%, more preferably at least 85%, even more preferably at least 90%, even more preferably at least 95% and most preferably at least 99% identity to an amino acid sequence shown in SEQ ID NO.7; a polynucleotide sequence encoding the protein has at least 60%, preferably at least 65%, preferably at least 70%, more preferably at least 75%, more preferably at least 80%, more preferably at least 85%, even more preferably at least 90%, even more preferably at least 95% and most preferably at least 99% identity to a polynucleotide sequence shown in SEQ ID NO.8.

In a preferred embodiment of the present invention, when the recombinant RyR2 protein is the Handle domain protein derived from the natural RyR2 protein, an amino acid sequence of the protein has at least 60%, preferably at least 65%, preferably at least 70%, more preferably at least 75%, more preferably at least 80%, more preferably at least 85%, even more preferably at least 90%, even more preferably at least 95% and most preferably at least 99% identity to an amino acid sequence shown in SEQ ID NO.9; a polynucleotide sequence encoding the protein has at least 60%, preferably at least 65%, preferably at least 70%, more preferably at least 75%, more preferably at least 80%, more preferably at least 85%, even more preferably at least 90%, even more preferably at least 95% and most preferably at least 99% identity to a polynucleotide sequence shown in SEQ ID NO.10.

In a preferred embodiment of the present invention, when the recombinant RyR2 protein is the HD1 domain protein derived from the natural RyR2 protein, an amino acid sequence of the protein has at least 60%, preferably at least 65%, preferably at least 70%, more preferably at least 75%, more preferably at least 80%, more preferably at least 85%, even more preferably at least 90%, even more preferably at least 95% and most preferably at least 99% identity to an amino acid sequence shown in SEQ ID NO.11; a polynucleotide sequence encoding the protein has at least 60%, preferably at least 65%, preferably at least 70%, more preferably at least 75%, more preferably at least 80%, more preferably at least 85%, even more preferably at least 90%, even more preferably at least 95% and most preferably at least 99% identity to a polynucleotide sequence shown in SEQ ID NO.12.

In a preferred embodiment of the present invention, when the recombinant RyR2 protein is the HD2 domain protein derived from the natural RyR2 protein, an amino acid sequence of the protein has at least 60%, preferably at least 65%, preferably at least 70%, more preferably at least 75%, more preferably at least 80%, more preferably at least 85%, even more preferably at least 90%, even more preferably at least 95% and most preferably at least 99% identity to an amino acid sequence shown in SEQ ID NO.13; a polynucleotide sequence encoding the protein has at least 60%, preferably at least 65%, preferably at least 70%, more preferably at least 75%, more preferably at least 80%, more preferably at least 85%, even more preferably at least 90%, even more preferably at least 95% and most preferably at least 99% identity to a polynucleotide sequence shown in SEQ ID NO.14.

In a preferred embodiment of the present invention, when the recombinant RyR2 protein is the central domain protein derived from the natural RyR2 protein, an amino acid sequence of the protein has at least 60%, preferably at least 65%, preferably at least 70%, more preferably at least 75%, more preferably at least 80%, more preferably at least 85%, even more preferably at least 90%, even more preferably at least 95% and most preferably at least 99% identity to an amino acid sequence shown in SEQ ID NO.15; a polynucleotide sequence encoding the protein has at least 60%, preferably at least 65%, preferably at least 70%, more preferably at least 75%, more preferably at least 80%, more preferably at least 85%, even more preferably at least 90%, even more preferably at least 95% and most preferably at least 99% identity to a polynucleotide sequence shown in SEQ ID NO.16.

In a preferred embodiment of the present invention, when the recombinant RyR2 protein is the EF-hand domain protein derived from the natural RyR2 protein, an amino acid sequence of the protein has at least 60%, preferably at least 65%, preferably at least 70%, more preferably at least 75%, more preferably at least 80%, more preferably at least 85%, even more preferably at least 90%, even more preferably at least 95% and most preferably at least 99% identity to an amino acid sequence shown in SEQ ID NO.17; a polynucleotide sequence encoding the protein has at least 60%, preferably at least 65%, preferably at least 70%, more preferably at least 75%, more preferably at least 80%, more preferably at least 85%, even more preferably at least 90%, even more preferably at least 95% and most preferably at least 99% identity to a polynucleotide sequence shown in SEQ ID NO.18.

In a preferred embodiment of the present invention, when the recombinant RyR2 protein is the U-motif protein derived from the natural RyR2 protein, an amino acid sequence of the protein has at least 60%, preferably at least 65%, preferably at least 70%, more preferably at least 75%, more preferably at least 80%, more preferably at least 85%, even more preferably at least 90%, even more preferably at least 95% and most preferably at least 99% identity to an amino acid sequence shown in SEQ ID NO.19; a polynucleotide sequence encoding the protein has at least 60%, preferably at least 65%, preferably at least 70%, more preferably at least 75%, more preferably at least 80%, more preferably at least 85%, even more preferably at least 90%, even more preferably at least 95% and most preferably at least 99% identity to a polynucleotide sequence shown in SEQ ID NO.20.

In a preferred embodiment of the present invention, when the recombinant RyR2 protein is the P2 domain protein derived from the natural RyR2 protein, an amino acid sequence of the protein has at least 60%, preferably at least 65%, preferably at least 70%, more preferably at least 75%, more preferably at least 80%, more preferably at least 85%, even more preferably at least 90%, even more preferably at least 95% and most preferably at least 99% identity to an amino acid sequence shown in SEQ ID NO.21; a polynucleotide sequence encoding the protein has at least 60%, preferably at least 65%, preferably at least 70%, more preferably at least 75%, more preferably at least 80%, more preferably at least 85%, even more preferably at least 90%, even more preferably at least 95% and most preferably at least 99% identity to a polynucleotide sequence shown in SEQ ID NO.22.

In a preferred embodiment of the present invention, when the recombinant RyR2 protein is the P2 domain fragment protein-1 derived from the natural RyR2 protein, an amino acid sequence of the protein has at least 60%, preferably at least 65%, preferably at least 70%, more preferably at least 75%, more preferably at least 80%, more preferably at least 85%, even more preferably at least 90%, even more preferably at least 95% and most preferably at least 99% identity to an amino acid sequence shown in SEQ ID NO.23; a polynucleotide sequence encoding the protein has at least 60%, preferably at least 65%, preferably at least 70%, more preferably at least 75%, more preferably at least 80%, more preferably at least 85%, even more preferably at least 90%, even more preferably at least 95% and most preferably at least 99% identity to a polynucleotide sequence shown in SEQ ID NO.24.

In a preferred embodiment of the present invention, when the recombinant RyR2 protein is the P2 domain fragment protein-2 derived from the natural RyR2 protein, an amino acid sequence of the protein has at least 60%, preferably at least 65%, preferably at least 70%, more preferably at least 75%, more preferably at least 80%, more preferably at least 85%, even more preferably at least 90%, even more preferably at least 95% and most preferably at least 99% identity to an amino acid sequence shown in SEQ ID NO.25; a polynucleotide sequence encoding the protein has at least 60%, preferably at least 65%, preferably at least 70%, more preferably at least 75%, more preferably at least 80%, more preferably at least 85%, even more preferably at least 90%, even more preferably at least 95% and most preferably at least 99% identity to a polynucleotide sequence shown in SEQ ID NO.26.

It should be noted that in a preferred embodiment of the present invention, a fragment protein derived from a P2 domain of the natural RyR2 protein includes at least a P2 core peptide segment, and an amino acid sequence of the core peptide segment has at least 60%, preferably at least 65%, preferred at least 70%, more preferably at least 75%, more preferably at least 80%, more preferably at least 85%, even more preferably at least 90%, even more preferably at least 95% and most preferably at least 99% identity to an amino acid sequence shown in SEQ ID NO.27; a polynucleotide sequence encoding the protein has at least 60%, preferably at least 65%, preferably at least 70%, more preferably at least 75%, more preferably at least 80%, more preferably at least 85%, even more preferably at least 90%, even more preferably at least 95% and most preferably at least 99% identity to a polynucleotide sequence shown in SEQ ID NO.28. Preferably, the core peptide segment includes a RyR2 S2808 site and/or a RyR2 S2814 site.

In a preferred embodiment of the present invention, the recombinant RyR2 protein is a P2 mutant derived from the natural RyR2 protein, and an amino acid sequence of the protein has at least 60%, preferably at least 65%, preferably at least 70%, more preferably at least 75%, more preferably at least 80%, more preferably at least 85%, even more preferably at least 90%, even more preferably at least 95% and most preferably at least 99% identity to an amino acid sequence shown in SEQ ID NO.29; a polynucleotide sequence encoding the protein has at least 60%, preferably at least 65%, preferably at least 70%, more preferably at least 75%, more preferably at least 80%, more preferably at least 85%, even more preferably at least 90%, even more preferably at least 95% and most preferably at least 99% identity to a polynucleotide sequence shown in SEQ ID NO.30.

A third aspect of the present invention provides a delivery vector of a RyR2 gene coding the recombinant RyR2 protein, and the delivery vector can be recombined with the RyR2 gene and integrated into a genome of cardiomyocytes. In order to adapt to an expression of the RyR2 gene in cardiac tissues, a cardiac tissue-specific promoter is arranged on the delivery vector.

The vector is one selected from the group consisting of a plasmid vector, a cosmid vector, a phage vector (such as a λ phage, a filamentous phage vector), a viral vector, a virus-like particle and a bacterial spore. Preferably, the vector is the viral vector, and the viral vector is one selected from the group consisting of adenovirus vector, adeno-associated virus (AAV) vector, α-virus vector, herpes virus vector, measles virus vector, poxvirus vector, vesicular stomatitis virus vector, retroviral vector and lentiviral vector. Most preferably, the vector is the AAV vector, such as AAV6 and AAV9.

A suitable dosage of the viral vector is 5×10⁹-1×10¹⁵ tvp, a more preferred dosage is 1×10¹¹-1×10¹³ tvp, and a most preferred dosage is 1×10¹² tvp.

The expression of a RyR2 protein is controlled by the cardiac tissue-specific promoter, i.e., a preferred vector also includes the cardiac tissue-specific promoter. Preferably, the cardiac tissue-specific promoter is selected from but not limited to a cardiac actin enhancer/elongation factor 1 promoter and a cytomegolo-virus enhancer/myosin light chain ventricle 2 promoter.

A fourth aspect of the present invention provides an anti-heart failure medicament composition, the medicament composition includes two forms: one containing a recombinant RyR2 protein, and further including a medically acceptable excipient, a carrier or a diluent, and the other containing a RyR2 gene, and further including a delivery vector and a medicinal carrier.

In a preferred embodiment of the present invention, an intracellular level of the recombinant RyR2 protein is elevated in at least 30% of cells in individual cardiac tissues, and a number of cardiac cells specifically expressing a RyR2 protein will depend on an underlying disease condition. Preferably, in a treated cardiac region, the intracellular level of the RyR2 protein is elevated as described above.

The intracellular level of the recombinant RyR2 protein is elevated in the cells of the individual cardiac tissues, further mediating a reduction in a level of a chemical modification at a specific site of the endogenous RyR2 protein in the cells. Preferably, the modification is one selected from the group consisting of phosphorylation, nitrification, methylation and acetylation. Most preferably, the site is S2808 and/or S2814, and the modification is the phosphorylation.

In an embodiment of the present invention, the anti-heart failure medicament composition is administered orally, intravenously, intramucosally, intraarterially, intramuscularly, or intracoronally. Preferably, the anti-heart failure medicament composition is administered intravenously.

In a further embodiment of the present invention, the intracellular level of the recombinant RyR2 protein is elevated for at least 7 days, more preferably for at least 14 days, and even more preferably for at least 28 days. This result is preferably obtained as a result of a single administration or a repeated administration.

In the present invention, a subject individual is healthy, or has a heart disease, or has a risk for the heart disease. The mechanism of the heart disease is related to the steady-state destruction of calcium cycling in cardiomyocytes and/or the cardiomyocyte contractile dysfunction. In a preferred embodiment, the heart disease is selected from post-ischemic contractile dysfunction, preferably post-ischemic contractile right and/or left ventricular dysfunction and congestive heart failure, preferably compensated and/or decompensated congestive heart failure, cardiogenic shock, septic shock, myocardial infarction, cardiomyopathy, cardiac valve dysfunction and ventricular disease, etc. For example, the heart disease is acute/chronic right ventricular dysfunction. More preferably, the heart disease is primary/secondary cardiomyopathy. The primary cardiomyopathy is preferably derived from hereditary cardiomyopathy and cardiomyopathy caused by spontaneous mutations. The secondary cardiomyopathy is preferably derived from ischemic cardiomyopathy caused by free arteriosclerosis, dilated cardiomyopathy caused by infection or poisoning, hypertensive heart disease caused by pulmonary artery and/or arterial hypertension, and cardiac valve disease.

A fifth aspect of the present invention provides an application of a RyR2 protein, a recombinant RyR2 protein or a gene encoding the RyR2 protein and the recombinant RyR2 protein in preparing an anti-heart failure medicament.

Preferably, the anti-heart failure medicament is a medicament increasing an intracellular level of the RyR2 protein in cardiac tissues, or reducing a level of a chemical modification at a specific site of the RyR2 protein in cells.

Preferably, the specific site of the RyR2 protein is at least one selected from the group consisting of S2808 and S2814, and the chemical modification is one selected from the group consisting of phosphorylation, nitrification, methylation and acetylation.

Therefore, a sixth aspect of the present invention provides an application of a RyR2 protein, a recombinant RyR2 protein or a polynucleotide of a gene encoding the RyR2 protein and the recombinant RyR2 protein in treating a heart disease by enhancing cardiac function of a subject. The subject has a heart disease, or has a risk for the heart disease. In a treatment window, it is used to increase a concentration of the RyR2 protein in individual cardiomyocytes.

Preferably, the recombinant RyR2 protein or the polynucleotide encoding the recombinant RyR2 protein is used for treating the heart disease by enhancing cardiac contractility of an individual with or at a risk of developing the heart disease, and for increasing the concentration of the RyR2 protein in the individual cardiomyocytes within the treatment window.

The cardiac function may enhance muscle contractile function by improving cardiac muscle function, contractile performance and/or calcium processing capacity. In a preferred embodiment, the cardiac function is enhanced by at least 15%, preferably at least 25%, more preferably at least 35%, most preferably at least 45%, and most preferably at least 50% compared to a control group.

Preferably, the control group is set as muscle function, contractile performance and/or calcium processing capacity of healthy volunteers, or an average value of a group of healthy volunteers. In a preferred embodiment, the cardiac function is enhanced by the recombinant RyR2 protein or the polynucleotide encoding the recombinant RyR2 protein, and the function exhibited by the RyR2 protein is at least one selected from the group consisting of an antiarrhythmia potential, an antiapoptosis potential, an ability to reduce a calcium spark frequency, an ability to prevent and/or reduce calcium leak from sarcoplasmic reticulum, and preferably an ability to restore a hemodynamic function in individuals with heart failure.

A seventh aspect of the present invention provides a method for increasing a concentration of a RyR2 protein in cardiomyocytes. The concentration of the RyR2 protein in the cardiomyocytes is increased by using a recombinant vector, and the recombinant vector includes a polynucleotide encoding a recombinant RyR2 protein or the recombinant RyR2 protein.

The Advantages of the Present Invention

The present invention provides an application of a RyR2 protein or a recombinant RyR2 protein in preparing an anti-heart failure medicament. The recombinant RyR2 protein is derived from a natural RyR2 protein fragment or mutant. Experiments prove that the exogenous recombinant RyR2 protein has high expression levels in both normal small animal model and diseased small animal model, so that the left ventricular ejection fraction of the experimental animals is improved to different degrees compared with that of a control group. Meanwhile, the animals of disease model can reduce the ventricular tachyarrhythmias triggered by (3-adrenaline to different degrees, and the cardiac function of each treatment group has different degrees of recovery. Therefore, the recombinant RyR2 protein of the present invention can effectively relieve and treat heart failure, and can be applied in preparing a medicament for treating heart failure, and has broad clinical application prospects.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic diagram showing a construction of a viral vector;

FIG. 2 is a diagram showing expression levels of exogenous recombinant RyR2 proteins in different treatment groups of normal mice;

FIG. 3 is a diagram showing results of left ventricular ejection fraction in different treatment groups of normal mice;

FIG. 4 is a diagram showing expression levels of the exogenous recombinant RyR2 proteins in different treatment groups of disease model mice;

FIG. 5 is a diagram showing results of left ventricular ejection fractions in different treatment groups of disease model mice;

FIG. 6 is a diagram showing results of Crp/ATP ratio in different treatment groups of disease model mice; and

FIG. 7A is a diagram showing results of phosphorylation degrees at RyR2 2808 in different treatment groups of disease model mice; and

FIG. 7B is a diagram showing results of phosphorylation degrees at RyR2 2814 in different treatment groups of disease model mice.

DETAILED DESCRIPTION OF THE EMBODIMENTS

The following embodiments and experimental examples further illustrate the present invention, which should not be interpreted as a limitation of the present invention. The embodiments do not include detailed descriptions of conventional methods, such as methods used to construct vectors and plasmids, methods of inserting genes encoding proteins into such vectors and plasmids, or methods of introducing plasmids into host cells. Such methods are well known to those of ordinary skill in the art and have been described in numerous publications, including: Sambrook, J., Fritsch, E. F. and Maniais, T. (1989) Molecular Cloning: A Laboratory Manual, 2^(nd) edition, Cold spring Harbor Laboratory Press.

Embodiment 1. Effects of Myocardial-Specific Exogenous Expressions of RyR2 Proteins on Cardiac Functions of Normal Small Animal Model

In order to detect the enhancing effect of AAV9-mediated RyR2 cDNA myocardial gene delivery on cardiac function, normal C57B/6 mice aged 6 months are divided into a treatment group and a control group. The intracellular deliveries of all recombinant RyR2 proteins are carried out using pAAV vectors with FLAG tags added to the C-terminals to facilitate detection. The construction of a viral vector is shown in FIG. 1. The polynucleotide encoding the recombinant protein described below is the recombinant protein region of the vector as shown in FIG. 1, with an ATG initiation codon added at the 5′ end if necessary. The specific grouping is described as follows.

The control group (CTRL) is a virus-empty group; the rSPRY1 group is a group expressing a recombinant SPRY domain protein, i.e., a polynucleotide of the recombinant protein region of the vector shown in FIG. 1 is shown as SEQ ID NO.2. The group rP1 is a group expressing a recombinant P1 domain protein, i.e., a polynucleotide of the recombinant protein region of the vector shown in FIG. 1 is shown as SEQ ID No.4. The rSPRY2 group is a group expressing a recombinant SPRY2 domain protein, i.e., a polynucleotide of the recombinant protein region of the vector shown in FIG. 1 is shown as SEQ ID No.6. The rSPRY3 group is a group expressing a recombinant SPRY3 domain protein, i.e., a polynucleotide of the recombinant protein region of the vector shown in FIG. 1 is shown as SEQ ID No.8 The rHandle group is a group expressing a recombinant Handle domain protein, i.e., a polynucleotide of the recombinant protein region of the vector shown in FIG. 1 is shown as SEQ ID No.10. The rHD1 group is a group expressing a recombinant HD1 domain protein, i.e., a polynucleotide of the recombinant protein region of the vector shown in FIG. 1 is shown as SEQ ID No.12. The rHD2 group is a group expressing a recombinant HD2 domain protein, i.e., a polynucleotide of the recombinant protein region of the vector shown in FIG. 1 is shown as SEQ ID No.14. The rCentral domain group is a group expressing a recombinant central domain protein, i.e., a polynucleotide of the recombinant protein region of the vector shown in FIG. 1 is shown as SEQ ID No.16. The rEF-hand group is a group expressing an EF-hand domain protein, i.e., a polynucleotide of the recombinant protein region of the vector shown in FIG. 1 is shown as SEQ ID No.18. The rU-motif group is a group expressing a U-domain protein, i.e., a polynucleotide of the recombinant protein region of the vector shown in FIG. 1 is shown as SEQ ID No.20. The rP2 group is a group expressing a P2 domain protein, i.e., a polynucleotide of the recombinant protein region of the vector shown in FIG. 1 is shown as SEQ ID No.22. The rP2-peptide-1 group is a group expressing a P2 domain fragment-1, i.e., a polynucleotide of the recombinant protein region of the vector shown in FIG. 1 is shown as SEQ ID No.24. The rP2-peptide-2 group is a group expressing a P2 domain fragment-2, i.e., a polynucleotide of the recombinant protein region of the vector shown in FIG. 1 is shown as SEQ ID No.26. The rP2-Core-peptide group is a group expressing a P2 domain core fragment, i.e., a polynucleotide of the recombinant protein region of the vector shown in FIG. 1 is shown as SEQ ID No.28. The rP2-mut group is a group expressing a P2 domain mutant, i.e., a polynucleotide of the recombinant protein region of the vector shown in FIG. 1 is shown as SEQ ID No.30.

Specific detection techniques for viral cardiomyocyte infection and protein expression refer to the non-patent document Voelkers et al. Circ Res (2011) 108: 27-39. Method for evaluating cardiac function by echocardiography refers to the non-patent document Most et al. JCI (2004) 114: 1550-1563. Methods for constructing viral vectors and utilizing myocardial-specific promoters refer to the non-patent document Pleger et al. Science Translational Medicine (2011) 3, 92ra64. The injection dose of virus particles is 1×10¹² tvp (total virus particles; tvp). The expression levels of myocardial-exogenous RyR2 proteins are detected by ELSIA or Western Blot.

The expression levels of the myocardial-exogenous RyR2 proteins of mice in different groups are shown in FIG. 2. FIG. 2 shows that, compared with the control group, each exogenous recombinant RyR2 gene has a relative high expression level in myocardial tissue of mice.

The left ventricular ejection fractions (LVEF) of mice in different groups are shown in FIG. 3. The results show that the left ventricular ejection fraction of mice in each treatment group is also increased to various degrees compared with the control group, indicating that each exogenous recombinant RyR2 protein can improve the cardiac pumping capacity and help prevent heart failure.

Embodiment 2. Effects of Myocardial-Specific Exogenous Expressions of RyR2 Proteins on Cardiac Functions of Diseased Small Animal Model

Further, the enhancing effect of AAV9-mediated RyR2 cDNA myocardial gene delivery on cardiac function is evaluated in the disease model. The steps are described as follows.

Firstly, the 6-month-old C57B/6 mice model with myocardial infarction is established. The model is established using temporary occlusion of the left anterior coronary artery with reference to the non-patent document Brinks et al. Circ Res (2010) 107: 1140-1149. Then, the mice are divided into a treatment group and a control group, and the C-terminals of all the RyR2 proteins are added with FLAG tags to facilitate detection. The specific construction method is the same as that of embodiment 1. The techniques for viral cardiomyocyte infection and methods for constructing viral vectors and utilizing myocardial-specific promoters are the same as those of embodiment 1. The injection dosage of virus particles is 1×10¹² tvp (total virus particles; tpv). The expression levels of myocardial-exogenous RyR2 proteins are detected by ELSIA or Western Blot.

The expression levels of the myocardial-exogenous RyR2 proteins of disease model mice in different groups are shown in FIG. 4. FIG. 4 shows that, compared with the control group, each exogenous recombinant RyR2 gene has a relative high expression level in myocardial tissue of mice.

The left ventricular ejection fractions (LVEF) of disease model mice in different groups are shown in FIG. 5. The results show that the left ventricular ejection fraction of mice in each treatment group is increased to various degrees compared with the control group. The increase degree of the P2 domain fragment-1 recombinant protein is the largest, followed by the P2 domain recombinant protein, indicating that each exogenous recombinant RyR2 protein can improve the cardiac pumping capacity, has significant effect on the treatment of heart failure, and contributes to the recovery of cardiac function.

FIG. 6 shows the CRP/ATP ratio level of mice in each disease model treatment group. The results show that the cardiac function of mice in each disease model treatment group has different degrees of recovery, and the recovery degree of the treatment group of P2 domain recombinant protein is the largest, increased by 67%, corresponding to the results in FIG. 5.

FIGS. 7A-B show that S2808 and S2814 of mice in each disease model treatment group are detected by ELISA (Amino acid coding referring to literature: Shan J. et al, J Clin Invest. 2010 December; 120 (12): 4375-87).

The preferred embodiments of the present invention have been specifically described above, but the present invention is not limited to the embodiments. A person skilled in the art can also make various equivalent modifications or substitutions without violating the spirit of the present invention. These equivalent modifications or substitutions shall all fall within the scope of claims of the present application. 

What is claimed is:
 1. A method of using a RyR2 protein or a recombinant RyR2 protein in preparing an anti-heart failure medicament.
 2. The method according to claim 1, wherein the recombinant RyR2 protein is a RyR2 protein fragment or a RyR2 protein mutant, and a length of the RyR2 protein fragment or a length of the RyR2 protein mutant is not less than 15 consecutive amino acid residues.
 3. The method according to claim 2, wherein the RyR2 protein fragment is one selected from the group consisting of a SPRY1 domain protein derived from a natural RyR2 protein, a P1 domain protein derived from the natural RyR2 protein, a SPRY2 domain protein derived from the natural RyR2 protein, a SPRY3 domain protein derived from the natural RyR2 protein, a Handle domain protein derived from the natural RyR2 protein, an HD1 domain protein derived from the natural RyR2 protein, an HD2 domain protein derived from the natural RyR2 protein, a central domain protein derived from the natural RyR2 protein, an EF-hand domain protein derived from the natural RyR2 protein, a U-motif protein derived from the natural RyR2 protein, a P2 domain protein derived from the natural RyR2 protein, a P2 domain fragment protein-1 derived from the natural RyR2 protein and a P2 domain fragment protein-2 derived from the natural RyR2 protein; the RyR2 protein mutant is a P2 mutant derived from the natural RyR2 protein; an amino acid sequence of the SPRY1 domain protein has at least 60% identity to an amino acid sequence shown in SEQ ID NO.1; an amino acid sequence of the P1 domain protein has at least 60% identity to an amino acid sequence shown in SEQ ID NO.3; an amino acid sequence of the SPRY2 domain protein has at least 60% identity to an amino acid sequence shown in SEQ ID NO.5; an amino acid sequence of the SPRY3 domain protein has at least 60% identity to an amino acid sequence shown in SEQ ID NO.7; an amino acid sequence of the Handle domain protein has at least 60% identity to an amino acid sequence shown in SEQ ID NO.9; an amino acid sequence of the HD1 domain protein has at least 60% identity to an amino acid sequence shown in SEQ ID NO.11; an amino acid sequence of the HD2 domain protein has at least 60% identity to an amino acid sequence shown in SEQ ID NO.13; an amino acid sequence of the central domain protein has at least 60% identity to an amino acid sequence shown in SEQ ID NO.15; an amino acid sequence of the EF-hand domain protein has at least 60% identity to an amino acid sequence shown in SEQ ID NO.17; an amino acid sequence of the U-motif protein has at least 60% identity to an amino acid sequence shown in SEQ ID NO.19; an amino acid sequence of the P2 domain protein has at least 60% identity to an amino acid sequence shown in SEQ ID NO.21; an amino acid sequence of the P2 domain fragment protein-1 has at least 60% identity to an amino acid sequence shown in SEQ ID NO.23; an amino acid sequence of the P2 domain fragment protein-2 has at least 60% identity to an amino acid sequence shown in SEQ ID NO.25; an amino acid sequence of the P2 mutant derived from the natural RyR2 protein has at least 60% identity to an amino acid sequence shown in SEQ ID NO.29.
 4. The method according to claim 3, wherein a sequence of a gene encoding the SPRY1 domain protein has at least 60% identity to a sequence shown in SEQ ID NO.2; a sequence of a gene encoding the P1 domain protein has at least 60% identity to a sequence shown in SEQ ID NO.4; a sequence of a gene encoding the SPRY2 domain protein has at least 60% identity to a sequence shown in SEQ ID NO.6; a sequence of a gene encoding the SPRY3 domain protein has at least 60% identity to a sequence shown in SEQ ID NO.8; a sequence of a gene encoding the Handle domain protein has at least 60% identity to a sequence shown in SEQ ID NO.10; a sequence of a gene encoding the HD1 domain protein has at least 60% identity to a sequence shown in SEQ ID NO.12; a sequence of a gene encoding the HD2 domain protein has at least 60% identity to a sequence shown in SEQ ID NO.14; a sequence of a gene encoding the central domain protein has at least 60% identity to a sequence shown in SEQ ID NO.16; a sequence of a gene encoding the EF-hand domain protein has at least 60% identity to a sequence shown in SEQ ID NO.18; a sequence of a gene encoding the U-motif protein has at least 60% identity to a sequence shown in SEQ ID NO.20; a sequence of a gene encoding the P2 domain protein has at least 60% identity to a sequence shown in SEQ ID NO.22; a sequence of a gene encoding the P2 domain fragment protein-1 has at least 60% identity to a sequence shown in SEQ ID NO.24; a sequence of a gene encoding the P2 domain fragment protein-2 has at least 60% identity to a sequence shown in SEQ ID NO.26; a sequence of a gene encoding the P2 mutant derived from the natural RyR2 protein has at least 60% identity to a sequence shown in SEQ ID NO.30.
 5. The method according to claim 3, wherein the P2 domain protein, the P2 domain fragment protein-1 or the P2 domain fragment protein-2 comprises at least a P2 core peptide segment, and an amino acid sequence of the P2 core peptide segment has at least 60% identity to an amino acid sequence shown in SEQ ID NO.27; a sequence of a gene encoding the P2 core peptide segment has at least 60% identity to a sequence shown in SEQ ID NO.28; the P2 core peptide segment comprises at least one selected from the group consisting of a RyR2 S2808 site and a RyR2 S2814 site.
 6. A delivery vector of a gene encoding the RyR2 protein or the recombinant RyR2 protein according to claim 4, comprising a cardiac tissue-specific promoter, wherein the cardiac tissue-specific promoter is arranged on the delivery vector.
 7. The delivery vector according to claim 6, wherein the delivery vector is one selected from the group consisting of a plasmid vector, a cosmid vector, a phage vector and a viral vector; the viral vector is one selected from the group consisting of adenovirus vector, adeno-associated virus (AAV) vector, α-virus vector, herpes virus vector, measles virus vector, poxvirus vector, vesicular stomatitis virus vector, retroviral vector and lentiviral vector; the cardiac tissue-specific promoter is one selected from the group consisting of a cardiac actin enhancer/elongation factor 1 promoter and a cytomegolo-virus enhancer/myosin light chain ventricle 2 promoter.
 8. The method according to claim 1, wherein the anti-heart failure medicament is a medicament increasing an intracellular level of the RyR2 protein in cardiac tissues, or a medicament reducing a level of a chemical modification at a specific site of the RyR2 protein in cells.
 9. The method according to claim 1, wherein a specific site of the RyR2 protein is at least one selected from the group consisting of S2808 and S2814, and a chemical modification is one selected from the group consisting of phosphorylation, nitrification, methylation and acetylation.
 10. An anti-heart failure medicament composition comprising the RyR2 protein or the recombinant RyR2 protein according to claim 1, further comprising a medicinal excipient, a medicinal carrier or a medicinal diluent.
 11. An anti-heart failure medicament composition comprising a gene encoding the RyR2 protein or the recombinant RyR2 protein according to claim 4, further comprising a delivery vector, and a medicinal carrier, wherein the delivery vector comprises a cardiac tissue-specific promoter, and the cardiac tissue-specific promoter is arranged on the delivery vector.
 12. A method for increasing a concentration of a RyR2 protein in cardiomyocytes, wherein the concentration of the RyR2 protein in the cardiomyocytes is increased by using a recombinant vector, the recombinant vector comprises a polynucleotide or a recombinant RyR2 protein, and the polynucleotide encodes the recombinant RyR2 protein.
 13. The anti-heart failure medicament composition according to claim 10, wherein the recombinant RyR2 protein is a RyR2 protein fragment or a RyR2 protein mutant, and a length of the RyR2 protein fragment or a length of the RyR2 protein mutant is not less than 15 consecutive amino acid residues.
 14. The anti-heart failure medicament composition according to claim 13, wherein the RyR2 protein fragment is one selected from the group consisting of a SPRY1 domain protein derived from a natural RyR2 protein, a P1 domain protein derived from the natural RyR2 protein, a SPRY2 domain protein derived from the natural RyR2 protein, a SPRY3 domain protein derived from the natural RyR2 protein, a Handle domain protein derived from the natural RyR2 protein, an HD1 domain protein derived from the natural RyR2 protein, an HD2 domain protein derived from the natural RyR2 protein, a central domain protein derived from the natural RyR2 protein, an EF-hand domain protein derived from the natural RyR2 protein, a U-motif protein derived from the natural RyR2 protein, a P2 domain protein derived from the natural RyR2 protein, a P2 domain fragment protein-1 derived from the natural RyR2 protein and a P2 domain fragment protein-2 derived from the natural RyR2 protein; the RyR2 protein mutant is a P2 mutant derived from the natural RyR2 protein; an amino acid sequence of the SPRY1 domain protein has at least 60% identity to an amino acid sequence shown in SEQ ID NO.1; an amino acid sequence of the P1 domain protein has at least 60% identity to an amino acid sequence shown in SEQ ID NO.3; an amino acid sequence of the SPRY2 domain protein has at least 60% identity to an amino acid sequence shown in SEQ ID NO.5; an amino acid sequence of the SPRY3 domain protein has at least 60% identity to an amino acid sequence shown in SEQ ID NO.7; an amino acid sequence of the Handle domain protein has at least 60% identity to an amino acid sequence shown in SEQ ID NO.9; an amino acid sequence of the HD1 domain protein has at least 60% identity to an amino acid sequence shown in SEQ ID NO.11; an amino acid sequence of the HD2 domain protein has at least 60% identity to an amino acid sequence shown in SEQ ID NO.13; an amino acid sequence of the central domain protein has at least 60% identity to an amino acid sequence shown in SEQ ID NO.15; an amino acid sequence of the EF-hand domain protein has at least 60% identity to an amino acid sequence shown in SEQ ID NO.17; an amino acid sequence of the U-motif protein has at least 60% identity to an amino acid sequence shown in SEQ ID NO.19; an amino acid sequence of the P2 domain protein has at least 60% identity to an amino acid sequence shown in SEQ ID NO.21; an amino acid sequence of the P2 domain fragment protein-1 has at least 60% identity to an amino acid sequence shown in SEQ ID NO.23; an amino acid sequence of the P2 domain fragment protein-2 has at least 60% identity to an amino acid sequence shown in SEQ ID NO.25; an amino acid sequence of the P2 mutant derived from the natural RyR2 protein has at least 60% identity to an amino acid sequence shown in SEQ ID NO.29.
 15. The anti-heart failure medicament composition according to claim 11, wherein the delivery vector is one selected from the group consisting of a plasmid vector, a cosmid vector, a phage vector and a viral vector; the viral vector is one selected from the group consisting of adenovirus vector, adeno-associated virus (AAV) vector, α-virus vector, herpes virus vector, measles virus vector, poxvirus vector, vesicular stomatitis virus vector, retroviral vector and lentiviral vector; the cardiac tissue-specific promoter is one selected from the group consisting of a cardiac actin enhancer/elongation factor 1 promoter and a cytomegolo-virus enhancer/myosin light chain ventricle 2 promoter. 